1. Field of the Invention
The present invention relates to an antifungal polypeptide, AlyAFP, obtainable from flowers of plants in the genus Alyssum, and methods for controlling pathogenic fungi employing this antifungal polypeptide. The antifungal polypeptide can be applied directly to a plant, applied to a plant in the form of microorganisms that produce the polypeptide, or plants themselves can be genetically modified to produce the polypeptide. The present invention also relates to DNA sequences, microorganisms, plants, and compositions useful in these methods.
2. Description of Related Art
A number of plant polypeptides and proteins exhibiting antifungal activity against a variety of plant pathogenic fungi have been isolated (Bowles (1990) Annu. Rev. Biochem. 59:873-907; Brears et al. (1994) Agro-Food-Industry Hi-Tech. 10-13). These antifungal polypeptides and proteins, encompassing several classes including chitinases, cysteine-rich chitin-binding proteins, xcex2-1,3-glucanases, permatins (including zeamatins), thionins, ribosome-inactivating proteins, and non-specific lipid transfer proteins, are believed to play important roles in plant defense against fungal infection.
Recently, another group of plant proteins has been found to function as defensins in combatting infections by plant pathogens (PCT International Publication WO 93/05153). Two small cysteine-rich proteins isolated from radish seed, Rs-AFP1 and Rs-AFP2, were found to inhibit the growth of many pathogenic fungi when the pure protein was added to an in vitro antifungal assay medium. Transgenic tobacco plants containing the gene encoding Rs-AFP2 protein were found to be more resistant to attack by fungi than non-transformed plants.
Proteins similar to radish seed Rs-AFP2 have been isolated from seeds of many other plants (PCT International Publication WO 93/05153; Broekaert et al. (1995) Plant Physiol. 108:1353-1358). All the proteins in this group share similarity in their amino acid sequence, but differ in their antifungal activities against various fungi, especially in the presence of different mono- and divalent salts in the assay medium, which more closely resembles the physiological condition in plant cells: the antifungal activity of some antifungal proteins is dramatically reduced in the presence of 1 mM CaCl2 and 50 mM KCl (Terras et al. (1992) J. Biol. Chem. 267:15301-15309). The usefulness of an antifungal protein for genetically engineering plant disease resistance can be greatly influenced by the sensitivity of the antifungal activity to salt concentration, since metal ions such K+, Na+, Ca2+, and Mg2+ are required for normal physiological functions and are therefore abundantly present in plant cells.
The use of natural protein products to control plant pathogens has been demonstrated, for example, in European Patent Application 0 392 225.
The present inventors have discovered a new polypeptide, AlyAFP, exhibiting broad spectrum antifungal activity against plant pathogenic and other fungi. In one aspect, the present invention provides an isolated antifungal polypeptide comprising the amino acid sequence shown in SEQ ID NO:2, and biologically functional equivalents thereof.
AlyAFP, or biologically functional equivalents thereof, can be isolated from plants, or produced or synthesized by any suitable method known in the art, including direct chemical synthesis, synthesis in heterologous biological systems such as microbial, plant, and animal systems, tissue cultures, cell cultures, or in vitro translation systems.
The present invention also provides isolated DNA sequences encoding the antifungal polypeptides of the present invention, and genetic constructs and methods for inserting such DNA sequences into host cells for the production of the polypeptides encoded thereby.
The present invention also provides microorganisms and plants transformed with DNA nucleotide sequences encoding the antifungal polypeptides according to the present invention.
The present invention provides transformed plants that express antifungal polypeptides according to the invention, as well as plants that co-express these antifungal polypeptides along with other antifungal, antibacterial, or antiviral pathogenesis-related peptides, polypeptides, or proteins; insecticidal proteins, e.g., Bacillus thuringiensis (B.t.) proteins; and proteins involved in improving the quality of plant products or agronomic performance of plants. Simultaneous co-expression of multiple antifungal proteins in plants is advantageous in that it exploits more than one mode of action to control fungal damage. This can minimize the possibility of developing resistant fungal strains, broaden the scope of resistance, and potentially result in a syngergistic antifungal effect, thereby enhancing the level of resistance. Note WO 92/17591, for example, in this regard.
Examples of plants transformed to express B.t. genes are disclosed in European Patent Publication 0 385 962, which corresponds to U.S. Ser. No. 07/476,661, filed Feb. 12, 1990, by Fischhoff et al.
Non-limiting examples of DNAs that can be co-expressed along with DNAs encoding the polypeptides of the present invention include 1) DNAs encoding enzymes such as: glucose oxidase (which converts glucose to gluconic acid, concomitantly producing hydrogen peroxide which confers broad spectrum resistance to plant pathogens); pyruvate oxidase; oxylate oxidase; cholesterol oxidase; amino acid oxidases; and other oxidases that use molecular oxygen as a primary or secondary substrate to produce peroxides, including hydrogen peroxide; 2) pathogenesis related proteins such as SAR8.2a and SARB.2b proteins; the acidic and basic forms of tobacco PR-1a, PR-1b, PR-1c, PR-1xe2x80x2, PR-2, PR-3, PR-4, PR-5, PR-N, PR-O, PR-Oxe2x80x2, PR-P, PR-Q, PR-S, and PR-R proteins; chitinases such as tobacco basic chitinase and cucumber chitinase/lysozyme; peroxidases such as cucumber basic peroxidase; glucanases such as tobacco basic glucanase; osmotin-like proteins; 3) viral capsid proteins and replicases of plant viruses; 4) plant R-genes (resistance genes), such as Arabidopsis RPS2 (Bent et al. (1994) Science 265:1856-1860), Arabidopsis RPM1 (Grant et al. (1995) Science 269:843-846), tobacco N-gene and Nxe2x80x2-gene (Whitham et al. (1994) Cell 78:1101-1115), tomato Cf-9 (Jones et al. (1994) Science 266:789-793), flax L6 (Ellis et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92: 4185), and rice Xa21 (Song et al. (1995) Science 270: 1804-1806). These genes can be expressed using constitutive promoters, tissue-specific promoters, or promoters inducible by fungal pathogens or other biological or chemical inducers; 5) pathogen Avr genes, such as Cladosporium fulvum Avr9 (Van Den Ackerveken et al. (1992) Plant J. 2:359), that can be expressed using pathogen- or chemical-inducible promoters; and 6) genes that are involved in the biosynthesis of salicylic acid, such as benzoic acid 2-hydroxylase (Leon et al. (1995) Proc. Natl. Acad. Sci. USA 92:10413-10417).
A number of publications have discussed the use of plant and bacterial glucanases, chitinases, and lysozymes to produce transgenic plants exhibiting increased resistance to various microorganisms such as fungi. These include EP 0 292 435, EP 0 290 123, WO 88/00976, U.S. Pat. No. 4,940,840, WO 90/07001, EP 0 392 225, EP 0 307 841, EP 0 332 104, EP 0 440 304, EP 0 418 695, EP 0 448 511, and WO 91/06312. The use of osmotin-like proteins is discussed in WO 91/18984.
In accomplishing the foregoing, there is provided in accordance with various aspects of the present invention:
An isolated polypeptide comprising the amino acid sequence shown in SEQ ID NO:2.
An isolated DNA molecule encoding this isolated polypeptide. The isolated DNA molecule can be a cDNA molecule comprising the nucleotide sequence shown in SEQ ID NO:12. Alternatively, this cDNA molecule can comprise nucleotides 116 to 269 of the nucleotide sequence shown in SEQ ID NO:12.
A recombinant, double-stranded DNA molecule, comprising operatively linked in the 5xe2x80x2 to 3xe2x80x2 direction:
a) a promoter that functions in plant cells to cause the production of an RNA sequence;
b) a structural coding sequence that encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO:2; and
c) a 3xe2x80x2 non-translated region that functions in plant cells to cause transcriptional termination and the addition of polyadenylate nucleotides to the 3xe2x80x2 end of said RNA sequence.
The structural coding sequence of the foregoing DNA molecule can be a cDNA molecule comprising the nucleotide sequence shown in SEQ ID NO:12, or nucleotides 116 to 269 of the nucleotide sequence shown in SEQ ID NO:12. Similar recombinant, double-stranded DNA molecules, containing appropriate promoters and other regulatory sequences, can be introduced into animal, fungal, and bacterial cells to obtain transformed cells expressing the structural coding sequence.
The promoter of the foregoing DNA molecule can be the FMV 35S promoter, the CaMV 35S promoter, the ssRUBISCO promoter, the eIF-4A promoter, the LTP promoter, the actin promoter, or the ubiquitin promoter.
A method of controlling fungal damage to a plant, comprising providing to the locus of said plant an isolated polypeptide comprising or consisting essentially of the amino acid sequence shown in SEQ ID NO:2. The fungal damage can be caused by a fungus selected from the group consisting of the genera Alternaria; Ascochyta; Botrytis; Cercospora; Colletotrichum; Diplodia; Erysiphe; Fusarium; Gaeumanomyces; Helminthosporium; Macrophomina; Nectria; Peronospora; Phoma; Phymatotrichum; Phytophthora; Plasmopara; Podosphaera; Puccinia; Puthium; Pyrenophora; Pyricularia; Pythium; Rhizoctonia; Scerotium; Sclerotinia; Septoria; Thielaviopsis; Uncinula; Venturia; and Verticillium. In this method, the polypeptide can be provided to the plant locus by plant-colonizing microorganisms which produce the antifungal polypeptide, by applying a composition comprising the isolated polypeptide thereto, or by expressing DNA encoding the polypeptide within cells of the plant.
A method of controlling fungal damage to a plant, comprising the steps of:
a) inserting into the genome of a plant cell a recombinant, double-stranded DNA molecule comprising:
(i) a promoter that functions in plant cells to cause the production of an RNA sequence;
(ii) a structural coding sequence that encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO:2;
(iii) a 3xe2x80x2 non-translated region that functions in said plant cells to cause transcriptional termination and the addition of polyadenylate nucleotides to the 3xe2x80x2 end of said RNA sequence;
b) obtaining transformed plant cells; and
c) regenerating from said transformed plant cells a genetically transformed plant, cells of which express an antifungal effective amount of said polypeptide.
In the foregoing method, the structural coding sequence can comprise the nucleotide sequence shown in SEQ ID NO:12, or nucleotides 116 to 269 of the nucleotide sequence shown in SEQ ID NO:12. The promoter can be the FMV 35S promoter, the CaMV 35S promoter, the ssRUBISCO promoter, the EIF-4A promoter, the LTP promoter, the actin promoter, or the ubiquitin promoter.
A plant, cells of which contain an antifungal effective amount of a polypeptide comprising the amino acid sequence shown in SEQ ID NO:2.
The foregoing plant can be produced by a method comprising the steps of:
a) inserting into the genome of a plant cell a recombinant, double-stranded DNA molecule comprising:
(i) a promoter that functions in plant cells to cause the production of an RNA sequence;
(ii) a structural coding sequence that encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NO:2;
(iii) a 3xe2x80x2 non-translated region that functions in said plant cells to cause transcriptional termination and the addition of polyadenylate nucleotides to the 3xe2x80x2 end of said RNA sequence;
b) obtaining transformed plant cells; and
c) regenerating from said transformed plant cells a genetically transformed plant, cells of which expresses an antifungal effective amount of said polypeptide.
The structural coding sequence employed in the foregoing method can comprise the nucleotide sequence shown in SEQ ID NO:12, or nucleotides 116 to 269 of the nucleotide sequence shown in SEQ ID NO:12.
Furthermore, the genome of this plant can comprise one or more additional DNA molecules encoding an antifungal peptide, polypeptide, or protein, wherein said one or more additional DNA molecules are expressed and produce an antifungal effective amount of said peptide, polypeptide, or protein encoded thereby. The additional DNA molecule can also comprise DNA encoding a B.t. endotoxin, wherein said DNA is expressed and produces an anti-insect effective amount of B.t. endotoxin. This plant can be a member selected from the group consisting of apple, barley, broccoli, cabbage, canola, carrot, citrus, corn, cotton, garlic, oat, onion, an ornamental plant, pea, peanut, pepper, potato, rice, rye, sorghum, soybean, strawberry, sugarbeet, sugarcane, tomato, a vine, and wheat. The present invention also encompasses a potato seedpiece produced by this plant.
A method of combatting an undesired fungus, comprising contacting the undesired fungus with an antifungal effective amount of an isolated polypeptide comprising the amino acid sequence shown in SEQ ID NO:2.
An antifungal composition, comprising an antifungal effective amount of an isolated polypeptide comprising the amino acid sequence shown in SEQ ID NO:2, and an acceptable carrier. The antifungal composition can be used for inhibiting the growth of, or killing, pathogenic fungi. These compositions can be formulated by conventional methods such as those described in, for example, Winnacker-Kuchler (1986) Chemical Technology, Fourth Edition, Volume 7, Hanser Verlag, Munich; van Falkenberg (1972-1973) Pesticide Formulations, Second Edition, Marcel Dekker, N.Y.; and K. Martens (1979) Spray Drying Handbook, Third Edition, G. Goodwin, Ltd., London. Necessary formulation aids, such as carriers, inert materials, surfactants, solvents, and other additives are also well known in the art, and are described, for example, in Watkins, Handbook of Insecticide Dust Diluents and Carriers, Second Edition, Darland Books, Caldwell, N.J., and Winnacker-Kuchler (1986) Chemical Technology, Fourth Edition, Volume 7, Hanser Verlag, Munich. Using these formulations, it is also possible to prepare mixtures of the present antifungal polypeptide with other pesticidally active substances, fertilizers and/or growth regulators, etc., in the form of finished formulations or tank mixes.
Antifungal compositions contemplated herein also include those in the form of host cells, such as bacterial and fungal cells, capable of the producing the present antifungal polypeptide. and which can colonize roots and/or leaves of plants. Examples of bacterial cells that can be used in this manner include strains of Agrobacterium, Arthrobacter, Azospyrillum, Clavibacter, Escherichia, Pseudomonas, Rhizobacterium, and the like.
Numerous conventional fungal antibiotics and chemical fungicides with which the present antifungal polypeptide can be combined are known in the art and are described in Worthington and Walker (1983) The Pesticide Manual, Seventh Edition, British Crop Protection Council. These include, for example, polyoxines, nikkomycines, carboxyamides, aromatic carbohydrates, carboxines, morpholines, inhibitors of sterol biosynthesis, and organophosphorus compounds. Other active ingredients which can be formulated in combination with the present antifungal polypeptide include, for example, insecticides, attractants, sterilizing agents, acaricides, nematocides, and herbicides. U.S. Pat. No. 5,421,839 contains a comprehensive summary of the many active agents with which substances such as the present antifungal polypeptide can be formulated.
Whether alone or in combination with other active agents, the antifungal polypeptide of the present invention should be applied at a concentration in the range of from about 0.1 xcexcg/ml to about 100 mg/ml, preferably between about 5 xcexcg/ml and about 5 mg/ml, at a pH in the range of from about 3.0 to about 9.0. Such compositions can be buffered using, for example, phosphate buffers between about 1 mM and 1 M, preferably between about 10 mM and 100 mM, more preferably between about 15 mM and 50 mM. In the case of low buffer concentrations, it is desirable to add a salt to increase the ionic strength, preferably NaCl in the range of from about 1 mM to about 1 M, more preferably about 10 mM to about 100 mM.
Further scope of the applicability of the present invention will become apparent from the detailed description and drawings provided below. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.